Escherichia coli ST224 and IncF/blaCTX-M-55 plasmids drive resistance to extended-spectrum cephalosporins in poultry flocks in Parana, Brazil.

Resistant Enterobacterales of avian intestinal origin can contaminate carcasses during broiler processing and thereby spread through the human food chain. This study aimed at assessing the prevalence, diversity and genomic characteristics of ESBL/AmpC Enterobacterales in poultry flocks from different farms and cities in the state of Paraná, Brazil. Enterobacterales isolated from cloacal samples were subjected to antimicrobial susceptibility testing (AST). ESBL/AmpC isolates were whole-genome sequenced and subjected to S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blotting to determine the location of resistant genes on plasmids. A surprisingly high proportion of E. coli (40.6 %) collected on non-selective plates presented an ESBL/AmpC phenotype. Multidrug resistance was statistically not higher in ESBL/AmpC E. coli having the potential to be Avian Pathogenic (APEC-like) compared to non-APEC-like ESBL/AmpC E. coli isolates. Resistance to antibiotics not authorized for use in poultry in the State of Paraná was observed, suggesting that antimicrobial resistance (AMR) is co-selected by the use of veterinary-licensed antibiotics. Phylogenetic analyzes revealed the presence of identical or highly similar ESBL/AmpC E. coli clones on farms distant up to 100 km of each other; this strongly suggests that the centralization and verticalization of the poultry industry can facilitate the spread of resistant bacteria among different farms, companies, and cities. The molecular characterization of clones and plasmids proved the dominance of the ST224 E. coli lineage and the IncF/blaCTX-M-55 plasmid, possibly indicating the emergence of successful clones and plasmids adapted to the chicken host. Our data contribute to the epidemiological tracking of resistance mechanisms in Enterobacterales from poultry and to knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.

Epidemic spread of IncI1/pST113 plasmid carrying the Extended-Spectrum Beta-Lactamase (ESBL) blaCTX-M-8 gene in Escherichia coli of Brazilian cattle.

INTRODUCTION: The prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide and the Agri-Food sector acts as a reservoir of clinically relevant ESBL genes. Our study aimed at detecting and characterizing ESBL-producing Enterobacteriaceae responsible for intestinal colonization of Brazilian bovines. MATERIAL AND METHODS: ESBL-producing E. coli isolates were recovered from fecal samples of healthy cattle in Northwest Brazil. Antimicrobial susceptibility was determined by disk diffusion. Resistance and virulence genes were identified by PCR and amplicons were sequenced, clonality was assessed by PFGE and MLST, and plasmids were characterized by replicon typing, S1-PFGE and Southern blot hybridizations. Transferability of ESBL genes was assessed by conjugation assay. RESULTS: A total of 40 ESBL-producing E. coli were characterized, which originated from 34/191 animals (17.8 %) and 15/22 farms (68.2 %). The blaCTX-M-8 gene was the most frequent ESBL gene (62.5 %), followed by blaSHV-2a (20.0 %), blaCTX-M-2 (10.0 %), and blaCTX-M-15 (7.5 %). The blaCTX-M-8 gene was localized on the IncI1/pST113 plasmid in multiple E. coli sequence types across unrelated animals and farms. DISCUSSION: We report the first characterization and a high prevalence of ESBL-producing E. coli in the beef cattle sector in Brazil, which is mainly supported by the spread of an epidemic IncI1/pST113/blaCTX-M-8 plasmid. Since Brazil is one of the biggest beef meat exporters worldwide, the spread of this ESBL plasmid across other sectors, countries and continents should be considered with attention.

Pandemic Escherichia coli ST648 isolate harbouring fosA3 and blaCTX-M-8 on an IncI1/ST113 plasmid: A new successful combination for the spread of fosfomycin resistance?

OBJECTIVES: The emergence of Enterobacteriaceae isolates resistant to the last-resort antibiotic fosfomycin outside of Asia is a public-health issue. Here we report the draft genome of an Escherichia coli isolate presenting both an extended-spectrum ß-lactamase (ESBL) and the fosA3 gene in a healthy cow in Brazil. METHODS: Whole genomic DNA from E. coli E12 was extracted and 2×150-bp paired-end reads were generated using Illumina sequencing technology. De novo genome assembly was performed using SPAdes v.3.11 and the draft genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline. Further analyses were performed using the Center for Genomic Epidemiology databases. RESULTS: The 5045934-bp genome displayed several resistance genes, including the fosA3 and blaCTX-M-8 genes. Southern blot experiments showed that they were co-located on an IncI1/ST113 plasmid. CONCLUSION: Presence of the fosA3 gene on the same common plasmid as blaCTX-M-8 will have to be monitored. This draft genome provides data that will help in tracing the dissemination of this gene and the evolution of its plasmidic support.

Extended-spectrum cephalosporin-resistant Escherichia coli isolated from chickens and chicken meat in Brazil is associated with rare and complex resistance plasmids and pandemic ST lineages.

Objectives: Brazil is the greatest exporter of chicken meat (CM) in the world. It is of utmost importance to monitor resistance to extended-spectrum cephalosporins (ESCs) in this sector because resistance to ESCs in Escherichia coli isolated from food-producing animals may contaminate humans through the food chain. Thus, the aim of this study was to characterize and compare ESC-resistant E. coli isolated from chickens and retail CM produced in south-eastern Brazil. Methods: Five CM samples and 117 chicken cloacal swabs (CCSs) were inoculated on MacConkey agar supplemented with cefotaxime. Presumptive E. coli colonies were identified and antimicrobial susceptibility was tested. Virulence and acquired blaESBL and blaAmpC genes were sought and genetic environments characterized. Isolates were typed by phylogenetic grouping, XbaI-PFGE and MLST. Results: All five CM samples and 36 CCSs (30.8%) were positive for the presence of ESC-resistant E. coli, leading to the selection of 58 resistant isolates. ESC resistance was mostly due to the presence of the chromosome-encoded blaCTX-M-2 gene, but plasmid-mediated blaCTX-M-2, blaCTX-M-8, blaCTX-M-15, blaCTX-M-55 and blaCMY-2 were also detected. Multireplicon plasmids were sporadically identified, such as IncHI2/P-blaCTX-M-2 and IncFII/N-blaCTX-M-55. Phylogroup D predominated, while PFGE and MLST revealed a high genetic diversity. Conclusions: Live Brazilian chickens and CM act as reservoirs of ESC-resistant E. coli and resistance genes are located on highly diverse genetic determinants. Potentially pathogenic strains, which may represent a threat to human health and a source of environmental contamination, were also identified. Active surveillance is therefore essential in Brazil's chicken production line.

Prevalence and molecular features of ESBL/pAmpC-producing Enterobacteriaceae in healthy and diseased companion animals in Brazil.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated AmpC (pAmpC)-carrying Enterobacteriaceae have widely disseminated in human, animal and environmental reservoirs. Pets have been recognized as a source of ESBL/pAmpC worldwide, and are possibly also a source of human contamination. The aim of this study was to document to what extent cats and dogs may act as a driving force in the spread of ESBLs and pAmpCs in Brazil. A total of 113 healthy stray cats and dogs and 74 sick pets were sampled, and extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R) were detected in 28/113 (24.8%) and 8/74 (10.8%) tested animals, respectively. Different Enterobacteriaceae isolates (mostly E. coli), a large number of E. coli clones (with ST90, ST457, ST973 and ST2541 being predominant), and several ESBL/pAmpC genes and plasmids were characterized, highlighting the ability of stray and pet cats and dogs to further spread a wide range of ESC-resistance determinants. The ESBL phenotype was due to the blaCTX-M-2 and blaCTX-M-8 genes, as found in human epidemiology in Brazil, but blaCTX-M-9 and blaCTX-M-15 were also identified. The pAmpC phenotype was systematically due to the presence of the blaCMY-2 gene, mostly carried by IncI1 ST12 plasmids. Our results showed that pets can be considered a significant reservoir of multidrug-resistant bacteria in Brazil. This is especially true for healthy stray dogs that displayed the highest prevalence (24.8%) of ESBLs/pAmpC resistance determinants, which can then be further spread both to the environment and to other animals or humans by contact.

Draft genome of a ST443 mcr-1- and blaCTX-M-2-carrying Escherichia coli from cattle in Brazil.

OBJECTIVES: Colistin is used in Brazil for the treatment of food-producing animals. The colistin resistance gene mcr-1 has already been reported from chicken and swine in this country. Here we report the draft genome of an Escherichia coli isolate presenting both an extended-spectrum ß-lactamase (ESBL) gene and the mcr-1 gene in a healthy cow in Brazil. METHODS: Whole genomic DNA from E. coli E12 was extracted and 2× 150-bp paired-end reads were generated using Illumina sequencing technology. De novo genome assembly was performed using SPAdes v.3.11 and the draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Further analyses were performed using Center for Genomic Epidemiology databases. Southern blots were performed to characterise plasmid location. RESULTS: The 5024393-bp genome displayed several resistance genes, including the mcr-1 and blaCTX-M-2 genes. These two genes were located on different plasmids (mcr-1 on an IncX4 plasmid and blaCTX-M-2 on an IncF plasmid). CONCLUSION: The genome sequence reported here can be compared with previously published genomes for mcr-1-producing isolates. This will ultimately help to understand the routes of dissemination of the resistance genes.

Draft genome sequence of a CTX-M-15-producing Escherichia coli ST345 from commercial chicken meat in Brazil.

Escherichia coli, the main host of the CTX-M-15 extended-spectrum ß-lactamase (ESBL) enzyme, is widely distributed and exchanged between the environment, animals and humans. Therefore, identification of blaCTX-M-15-positive lineages in food has a significant impact on public health. In this regard, until the end of 1990s, ESBL-producing isolates were mainly associated with hospital-acquired infections, with a predominance of SHV- and TEM-type enzymes. In recent years, a new trend has been observed among ESBL-producers, where most isolates now harbour CTX-M-type, being further isolated from community-acquired infections. Nowadays, CTX-M-15 has been recognised as the most important ESBL variant, invading virtually all human and animal compartments, leading to a global pandemic. Thus, whilst the rapid emergence and dissemination of CTX-M-15 among E. coli isolates has generated a large genetic reservoir from which other members of the Enterobacteriaceae family can easily acquire this resistance gene, there are an increasing number of new reservoirs and transmission mechanisms that must be investigated. In this study, we present the draft genome sequence of a CTX-M-15-producing E. coli ST345 isolated from commercial chicken meat in Brazil. This draft genome can be used as a reference sequence for comparative analysis among CTX-M-15-producers.